ANTIBACTERIAL POTENTIAL OF THE METHANOL STEM BARK EXTRACT OF INDICA

ORIGINAL ARTICLE,Vol-4 No.4

Asian Journal of Medical Science, Volume-4(2013) http://nepjol.info/index.php/AJMS 1Akuodor Godwin Chris tian, 2Udia Pius Monday, 3Udenze Emeka Charles , 4Ogbonna Onyemaechi John . Dept. of Pharmacology, College of Medical Sciences, University of Calabar, Nigeria.

ABSTRACT

CORRESPONDENCE : Objective: There is increasing need for potent antimicrobial agents to tackle Dr. Akuodor Godwin C the problem of diseases in man. In view of this, the activities of methanol Department of Pharmacology, stem bark extract of Stachytarpheta indica was evaluated against some College of Medical Sciences, disease causing microorganisms. University of Calabar, Nigeria Method : The activity of the extract against S. aureus, P.aeruginosa, E. coli, S. Email: typhi and Shigella spp was determined using agar diffusion technique. [email protected] Tel+2348036725237 Results: The methanol stem bark extract demonstrated significant activity against the test organisms. Th e Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of the stem bark extract ranged from 12-60 µg/ml.

“The search for new Conclusion: Further isolation of active compound responsible for the drugs to counter the challenges posed by antibacterial activity could be the potential sou rces of new antibacterial resistant strains of agents. bacteria might have started yielding results Key words: Antibacterial agent; Stachytarpheta indica ; Medicina ; as the investigation of Stem bark; Methanol; in vitro this plant has demonstrated enormous therapeutic potential.”

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INTRODUCTION Antibacterial resistance has become a global sedative, anti-fertility and rheumatism 10, 11 . In problem. Strategies to improve the current Northern Nigeria, a decoction of the leaves with situation include research in finding new natron is given for dysentery in human and for antibacterial agent of plant origin. have similar condition in horses 9.The study was designed great potential for producing new drugs of great to investigate the antibacterial activity of the benefit to mankind. Natural plants have been seen methanol stem bark extract of Stachytarpheta as a valuable source of medicinal agents with indica . The result is hoped to substantiate or proven potential for treating infectious diseases otherwise, the ethnomedicinal use of the stem with lesser side effects compared to synthetic bark extract for the treatment of bacteria and agents 1,2 . Many efforts have been made to discover possibly pave the way for its wider acceptability as new antimicrobial compounds from various kinds of antibacterial agent. sources such as animals, microorgan isms, soil and MATERIALS AND METHODS plants. One of such sou rces is folk medicine and systematic screening of them may result in the Plant collection discovery of novel effective The stem bark of Stachytarpheta indica was compounds 3, 4 .Despite the existence of potent collected from plants growing within the campus of antibiotic and antifungal agents, resistant or multi - University of Calabar, Nigeria. The plant material resistant strain s are continuously appearing; was authenticated by Mr. Frank I. Apejoye of the imposing the need for a permanent search and Department of Botany, University of Calabar, development of new antimicrobial agent of natural where a voucher spacemen (No.133) is maintained. origin 5, 6 . With increase in antibiotic resistance, The international plant name index is cost and inaccessibility (especially in rural areas) to Stachytarpheta indica Vahl. Enum.pl.1:206.1804. some orthodox modern a ntibiotics, medicinal The stem bark was cut into smaller pieces, dried at plants are fast gaining popularity even to urban and room temperature for 7 days and pulverized to dry civilized dwellers. There is an urgent need to powder using a mortar and pestle. evaluate the plants used in traditional medicine, Extraction of plant material with the aim of establishing their potential Five hundred grams (500 g) of the dry stem bark antibacterial effects and identifying t he compounds powder was extracted with methanol by cold responsible for their antimicrobial properties 7, 8 . maceration for 48h with constant shaking. The Stachytarpheta indica Vahl (Verbenaceae) is methanol extract was concentrated to dryness in vacuum at 40 oC. The yi eld was (12% w/w). The dry commonly known as snake weed. A well branched o herb, 2-3 ft high with very long narrow spikes; extract was stored in a refrigerator at 4 C until use flowers deep blue with white centre. The plant is for the experiment. known by various names in different parts of Phytochemical screening Nigeria, such as ‘Tsarkiyar kuse’ (Hausa), ‘Iru The methanol stem bark extract of Stachytarpheta amure’ (Yoruba) 9. It has been used locally as an indica was subjected to qualitative phytochemical 12, 13 abortifacient, and in the management of asthma, screening according to standard methods headache, alopecia, bronchitis, bruises, Test Organisms constipation, diarrhoea, skin sore, dysentery, Clinical isolates of Staphylococcus aureus, dysmenorrheal, fever, inflammation, liver disease, Pseudomonas aeruginosa, Salmonella typhi, poisoning ,tumor, venereal diseases, cataract, Escherichia coli and Shigella spp. obtained from

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Department of Microbiology, University of Calabar was a serial dilution of the extract to obtain Teaching Hospital, Calabar, Nigeria, were used for concentrations of 6.25, 12.5, 25, 50, 75 and 100 the experi ment. Purity plates of each bacterial µg/ml respectively. The test tube organism (0.5 ml) isolates were obtained by culturing on their was taken and transferred into each of the test respec tive selective media. Biochemical tests were tubes containing the mixture of the broth and the o performed to re-identify and confirm the identity extract and then incubated at 37 C for 24 h. The of the isolates. Fresh plates of the test organisms MIC was recorded as the least concentration of were made from the i solate cultures obtained on plant extract that completely inhibited the growth agar slants. Discrete colonies of fresh cultures of of the organism. different bacterial isolates were then picked and Determination of minimum bactericidal suspended in 5 ml nutrient broth in Bijou bottles concentration (MBC) and incubated for 24 h at 37 oC prior to The minimum bactericidal concentration of the antimicrobial susceptibility testing. plant extract on the clinical bacterial isolates was Determination of antibacterial activity carried out according to Nationa l Committee for The antibacterial activities of the methanol stem Clinical Laboratory Standard 18. A loopful of broth bark extract of Stachytarpheta indica was was collected from the determination of MIC tubes determined by agar well diffusion method of 14, 15, which did not show any growth and streaked on a 16 .Broth cultures of the test isolate (0.5 ml) sterile nutrient agar. All the plates were then containing 1x10 5 cfu/ml of organism was incubated at 37 oC for 24 h. The least conc entration introduced into sterile petri-dish and 15 ml of of the stem bark extract with no visible growth

Muller Hinton agar was added. The content was after incubation was taken as the minimum properly mixed and allowed to solidify. Holes were bactericidal concentration.

Statistical analysis bored on the plates, using a standard sterile cork borer of 6mm in diameter and the stem bark The mean of the reading measured for each zone extract reconstituted in distilled water at varying in the sensitivity test was taken to be the zone of concentrations of 25, 50, 75,100 and 125 µg/ml inhibiti on of the bacterial isolates. Two ways were transferred into the wells with the aid of ANOVA was used to compare means and micropipette. The experiments were carried out in differences were considered significant at p<0.05. duplicate. The plates were allowed to stand for 1 h for prediffusion of the extract to occur and RESULTS incubated at 37 o C for 24 h. At the end of the Phytochemical studies incubation, the diameter of the zones of inhibition Phytochemical screening of the stem bark extract were measured and recorded in mm. revealed the presence of saponins, flavonoids, Determination of minimum inhibi tory steroids, terpenoids, alkaloids, tannins and concentration (MIC) reducing sugars, while Phlobatannins and phenols The minimum inhibitory conce ntration was were not detected. These classes of compounds determined by the method as described by are reported to show important biological 19,20,21 Andrews 17 . Six sterile tubes were arranged in a activities (table 1). test tube rack and 0.5 ml of sterile nutrient broth Antibacterial activity of the stem bark extract was transferred into each test tube. Thereafter, The results of antibacterial activity of the methanol there

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stem bark extract of Stachytarpheta indica against showed good antimicrobial activity against the test organisms are shown in table 2. The zones pathogenic bacterial strains like Shigella spp, of inhibition of the isolates are a function of the Salmonella typhi, Pseudomonas aeruginosa antibacterial activity of the extract. The activity of Staphylococcus aureus and Escherichia coli . Plant the stem bark extract was shown to be based products have been effectively proven for concentration dependent. The extract showed their utilization as source for antimicrobial significant inhibit ion against all the test organisms. components. The fact t hat the methanol stem bark Distilled water used as respective controls were extract of Stachytarpheta indica showed activity inactive against the bacteria. against both gram-negative and gram-positive Minimum inhibitory concentration (MIC) and bacteria tested may indicate a broad spectrum of Minimum bactericidal Concentration activity 30 . This observation is very significant Table 3 shows the results of MIC and MBC because of the possibility of deve loping therapeutic determination on the test organisms. The lowest substances that will be active against multidrug - MIC and MBC of 12 and 25 µg/ml was resistant organisms. However, the very high demonstrated against S. aureus , while the MIC and significant antibacterial shown by the stem bark MBC values ranging between 15-60 µg/ml were extract of Stachytarpheta indica is beneficial as it demonstrated against the rest of the test indicates probably the emergence of a new organisms. antibiotic with such a wide spectrum of activity.

DISCUSSION The fact that treatment of infections caused by organisms such as Staphylococcus aureus, Plants ar e important source of potentially useful Pseudomonas aeruginosa, Salmonella typhi, structures for the development of new Escherichia coli and Shigella spp are increasingly chemotherapeutic agents. The first step towards becoming difficult fur ther strengthens the 22 this goal is the in vitro antibacterial activity assay . importance of these present findings and the need

Many reports are available on the antibacterial, for a continuous search for chemotherapeutic antiviral, antifungal, anthelmintics and anti - 23, 24, 25, 26,27. agents. Apart from antimicrobial activities, the stem inflammatory properties of plants Some bark extract of Stachytarpheta indica was found to of these observations have helped in identifying the possess analgesic, anti-inflammato ry and active principle responsible for such activities and in antipyretic activities 31. developing drugs for the therapeutic use in human. In conclusion, the present study has shown that the The phytoche mical investigation revealed the stem bark extract of Stachytarpheta indica presence of saponins, flavonoids, steroids, possesses antimicrobial properties; hence could be terpenoids, alkaloids, tannins and reducing sugars of considerable interest to the development of new (Table 1). These bioactive components which are drugs. The bioassay-guided fractionation and naturally occurring in most plant materials, are further characterization of the active principles known to be bactericidal, pesti cidal or fungicidal in 28,29 responsible for the antimicrobial potential of the nature . Thus, the presence of the above plant is underway in our laboratory. phytochemical components may account for the Conflict of interest statement high antimicrobial activity of the stem bark extract We declare that we have no conflict of interest. against some selected microorganisms. The methanol stem bark extract

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Table 1. Phytochemical constituents of the methanol stem bark extract of Stachytarpheta indica

Test Methanol extract Saponins ++ Flavonoids ++ Steroids ++ Tannins +++ Terpenoids ++ Alkaloids + Reducing sugars +++ Phlobatannins - Phenols - + = Slight presence; ++ = Medium presence; +++ = Heavy presence; - = Absence

Table2. Antibacterial potency of the methanol stem bark extract of Stachytarpheta indica (mm) Dose (µg/ml) Shigella spp. S. typhi S. aerus P. aeruginosa E. coli 12.5 9.53±3.0 7.53±3.2 13.0±4.7 9.78±6.0 9.0±3.0 25 11.56±3.3 9.81±5.0 17.8±5.9 16.63±4.6 11.9±5.0 50 15.63±6.4 13.63±4.0 22.13±5.8 17.0±0.0 13.0±5.0 75 18.50±2 .8 17.50±2.5 25.0±0.0 17.50±4.9 16.63±5.0 100 22.75±3.9 22.0±4.4 27.74±2.0 20.50±0.0 19.25±1.8 125 33.50±5.3 32.50±4.0 47.0±3.0 29.50±0.0 29.50±0.0 Values are shown in mean ± SEM

Table 3. Antibacterial effect (MIC and MBC in µg/ml) of methanol stem bark extract of Stachytarpheta indica Test organisms MIC MBC Staphylococcus aureus 20 40 Salmonella typhi 12 25 Shigella spp. 15 30 Pseudomonas aeruginosa 20 40 Esche richia coli 30 60

ACKNOWLEDGEMENT The authors are grateful to Mr Frank I. Apejoye of 2. Farnsworth NR, Loub WD. Information gathering and data bases that are pertinent to development of plant-derived the Department of Botany, University of Calabar, drugs in plants: The potentials for Extracting Protein, Nigeria for his botanical assistance. Medicines, and Other Useful Chemicals. Workshop Proceedings. OTA-BP-F-23. U.S. Congress, Office of REFERENCES Technology Assessment, Washington, D. C. 1983; pp. 178 - 179. 1. Akuodor GC, Essien AD, Ibrahim JA, Bassey A, Akpan JL, 3. Janovska D, Kubikova K, Kokoska L. Screening for Ikoro NC,Onyewenjo SC. Phytochemical and antimicrobial antimicrobial activity of some medicinal plant of properties of the methanolic extract of Bombax traditional Chinese medicine. Czech. J. Food Sci. 2003; 21: buonopozense leaf and root. Asian Journal of Medical Sciences. 2011; 2:190-194. 107-111.

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